Plant regulatory elements and methods of use thereof

ABSTRACT

The present disclosure provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions include a novel nucleotide sequence for regulatory elements from  Eupatorium  Vein Clearing Virus. A method for expressing a heterologous nucleotide sequence in a plant using the regulatory element sequences disclosed herein is provided. The method comprises transforming a plant or plant cell with a nucleotide sequence operably linked to one of the regulatory elements of the present disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Application NumberPCT/US2016/037857 filed Jun. 16, 2016, which claims the benefit of U.S.Provisional Application No. 62/231,155, filed Jun. 17, 2015, which ishereby incorporated herein in its entirety by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

A sequence listing having the file name “5971USPSP2_SequenceListing.TXT” created on May 22, 2014, and having a sizeof 5.6 kilobytes is filed in computer readable form concurrently withthe specification. The sequence listing is part of the specification andis herein incorporated by reference in its entirety.

FIELD

The present disclosure relates to the field of plant molecular biology,more particularly to regulation of gene expression in plants.

BACKGROUND

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of an operably linked regulatory element that isfunctional within the plant host. Choice of the regulatory elementsequence will determine when and where within the organism theheterologous DNA sequence is expressed. Where expression in specifictissues or organs is desired, tissue-preferred regulatory elements maybe used. Where gene expression in response to a stimulus is desired,inducible regulatory elements are the regulatory element of choice. Incontrast, where continuous expression is desired throughout the cells ofa plant, constitutive promoters are utilized. Additional regulatorysequences upstream and/or downstream from the core regulatory elementsequence may be included in the expression constructs of transformationvectors to bring about varying levels of expression of heterologousnucleotide sequences in a transgenic plant.

Frequently it is desirable to express a DNA sequence constitutively in aplant. For example, increased resistance of a plant to infection bysoil- and air-borne pathogens might be accomplished by geneticmanipulation of the plant's genome to comprise a constitutive regulatoryelement operably linked to a heterologous pathogen-resistance gene suchthat pathogen-resistance proteins are produced in the desired planttissue.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a constitutive promoter operably linked to anantisense nucleotide sequence, such that expression of the antisensesequence produces an RNA transcript that interferes with translation ofthe mRNA of the native DNA sequence.

Genetically altering plants through the use of genetic engineeringtechniques and thus producing a plant with useful traits requires theavailability of a variety of promoters. An accumulation of promoterswould enable the investigator to design recombinant DNA molecules thatare capable of being expressed at desired levels and cellular locales.Therefore, a collection of constitutive promoters would allow for a newtrait to be expressed at the desired level in the desired tissue. Thus,isolation and characterization of constitutive regulatory elements thatmay serve as regulatory regions for expression of heterologousnucleotide sequences of interest in a measured constitutive manner areneeded for genetic manipulation of plants.

SUMMARY

Compositions and methods for regulating expression of a heterologousnucleotide sequence of interest in a plant or plant cell are provided.DNA molecules comprising novel nucleotide sequences for regulatoryelements that initiate transcription are provided. In some embodimentsthe regulatory element has promoter activity initiating transcription inthe plant cell. Embodiments of the disclosure comprise the nucleic acidsequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a complement thereof, a nucleotidesequence comprising at least 20 contiguous nucleotides of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,wherein said sequence initiates transcription in a plant cell, and anucleotide sequence comprising a sequence having at least 85% sequenceidentity to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, wherein said sequenceinitiates transcription in the plant cell.

A method for expressing a heterologous nucleotide sequence in a plant orplant cell is provided. The method comprises introducing into a plant ora plant cell an expression cassette comprising a heterologous nucleotidesequence of interest operably linked to one of the regulatory elementsof the present disclosure. In this manner, the regulatory elementsequences are useful for controlling the expression of the operablylinked heterologous nucleotide sequence.

Further provided is a method for expressing a nucleotide sequence ofinterest in a constitutive manner in a plant. The method comprisesintroducing into a plant cell an expression cassette comprising aregulatory element of the disclosure operably linked to a heterologousnucleotide sequence of interest.

Expression of the nucleotide sequence of interest may provide formodification of the phenotype of the plant. Such modification includesmodulating the production of an endogenous product, as to amount,relative distribution, or the like, or production of an exogenousexpression product to provide for a novel function or product in theplant. In specific methods and compositions, the heterologous nucleotidesequence of interest comprises a gene product that confers herbicideresistance, pathogen resistance, insect resistance, and/or alteredtolerance to salt, cold, or drought.

Expression cassettes comprising the promoter sequences of the disclosureoperably linked to a heterologous nucleotide sequence of interest areprovided. Additionally provided are transformed plant cells, planttissues, seeds, and plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the Eupatorium Vein Clearing Virus (EVCV)regulatory region and truncations of the regulatory region. The 1104base pair EVCV regulatory region (FL) consists of a portion of the longintergenic region (LIR) upstream of a short ORF and stem loop structure.The sequence extends 5′ into the 3′ end of the last ORF of the EVCVgenome. The regulatory region was truncated (TR) from the 5′ end tosegments that consisted of 821 bp, 514 bp, 431 bp, 283 bp, and 116 bp.The position of the putative TATA box is depicted by the arrow.

FIG. 2 shows the nucleic acid sequence of the 1104 base pair full-lengthEVCV regulatory element (SEQ ID NO: 1). The putative TATA box isunderlined. Also indicated by inserted arrows are the 5′ ends of thetruncated EVCV regulatory elements as represented by the nucleic acidsequence of the 821 base pair truncated regulatory element EVCV TR1 (SEQID NO: 2), the nucleic acid sequence of the 514 base pair truncatedregulatory element EVCV TR2 (SEQ ID NO: 3), the nucleic acid sequence ofthe 431 base pair truncated regulatory element EVCV TR3 (SEQ ID NO: 4,the nucleic acid sequence of the 283 base pair truncated regulatoryelement EVCV TR4 (SEQ ID NO: 5) and the nucleic acid sequence of the 116base pair truncated regulatory element EVCV TR5 (SEQ ID NO: 6).

DETAILED DESCRIPTION

The disclosure relates to compositions and methods drawn to plantpromoters and methods of their use. The compositions comprise nucleotidesequences for the regulatory region of Eupatorium Vein Clearing Virus(EVCV). The compositions further comprise DNA constructs comprising anucleotide sequence for the regulatory region of EVCV operably linked toa heterologous nucleotide sequence of interest. In particular, thepresent disclosure provides for isolated nucleic acid moleculescomprising the nucleotide sequence set forth in SEQ ID NO: 1, andfragments, variants, and complements thereof.

The EVCV regulatory element sequences of the present disclosure includenucleotide constructs that allow initiation of transcription in a plant.In specific embodiments, the EVCV regulatory element sequence allowsinitiation of transcription in a constitutive manner. Such constructs ofthe disclosure comprise regulated transcription initiation regionsassociated with plant developmental regulation. Thus, the compositionsof the present disclosure include DNA constructs comprising a nucleotidesequence of interest operably linked to the EVCV regulatory elementsequence. One source for the EVCV regulatory region sequence is setforth in SEQ ID NO: 1.

Compositions of the disclosure include the nucleotide sequences for EVCVregulatory elements and fragments and variants thereof. In specificembodiments, the regulatory element sequences of the disclosure areuseful for expressing sequences of interest in a constitutive manner.The nucleotide sequences of the disclosure also find use in theconstruction of expression vectors for subsequent expression of aheterologous nucleotide sequence in a plant of interest or as probes forthe isolation of other EVCV-like regulatory elements.

Regulatory Elements

A regulatory element is a nucleic acid molecule having gene regulatoryactivity, i.e. one that has the ability to affect the transcriptionand/or translation of an operably linked transcribable polynucleotidemolecule. The term “gene regulatory activity” thus refers to the abilityto affect the expression of an operably linked transcribablepolynucleotide molecule by affecting the transcription and/ortranslation of that operably linked transcribable polynucleotidemolecule. Gene regulatory activity may be positive and/or negative andthe effect may be characterized by its temporal, spatial, developmental,tissue, environmental, physiological, pathological, cell cycle, and/orchemically responsive qualities as well as by quantitative orqualitative indications.

Regulatory elements such as promoters, leaders, introns, andtranscription termination regions are nucleic acid molecules that havegene regulatory activity and play an integral part in the overallexpression of genes in living cells. The term “regulatory element”refers to a nucleic acid molecule having gene regulatory activity, i.e.one that has the ability to affect the transcription and/or translationof an operably linked transcribable polynucleotide molecule. Isolatedregulatory elements, such as promoters and leaders that function inplants are therefore useful for modifying plant phenotypes through themethods of genetic engineering.

Regulatory elements may be characterized by their expression pattern,i.e. as constitutive and/or by their temporal, spatial, developmental,tissue, environmental, physiological, pathological, cell cycle, and/orchemically responsive expression pattern, and any combination thereof,as well as by quantitative or qualitative indications. A promoter isuseful as a regulatory element for modulating the expression of anoperably linked transcribable polynucleotide molecule.

As used herein, a “gene expression pattern” is any pattern oftranscription of an operably linked nucleic acid molecule into atranscribed RNA molecule. Expression may be characterized by itstemporal, spatial, developmental, tissue, environmental, physiological,pathological, cell cycle, and/or chemically responsive qualities as wellas by quantitative or qualitative indications. The transcribed RNAmolecule may be translated to produce a protein molecule or may providean antisense or other regulatory RNA molecule, such as a dsRNA, a tRNA,an rRNA, a miRNA, and the like.

As used herein, the term “protein expression” is any pattern oftranslation of a transcribed RNA molecule into a protein molecule.Protein expression may be characterized by its temporal, spatial,developmental, or morphological qualities as well as by quantitative orqualitative indications.

As used herein, the term “promoter” refers generally to a nucleic acidmolecule that is involved in recognition and binding of RNA polymeraseII and other proteins (trans-acting transcription factors) to initiatetranscription. A promoter may be initially isolated from the 5′untranslated region (5′ UTR) of a genomic copy of a gene. Alternately,promoters may be synthetically produced or manipulated DNA molecules.Promoters may also be chimeric, that is a promoter produced through thefusion of two or more heterologous DNA molecules.

In one embodiment, fragments are provided of a promoter sequencedisclosed herein. Promoter fragments may exhibit promoter activity, andmay be useful alone or in combination with other promoters and promoterfragments, such as in constructing chimeric promoters. In specificembodiments, fragments of a promoter are provided comprising at leastabout 50, 95, 150, 250, 500, or about 750 contiguous nucleotides of apolynucleotide molecule having promoter activity disclosed herein. Suchfragments may exhibit at least about 85 percent, about 90 percent, about95 percent, about 98 percent, or about 99 percent, or greater, identitywith a reference sequence when optimally aligned to the referencesequence.

A promoter or promoter fragment may also be analyzed for the presence ofknown promoter elements, i.e. DNA sequence characteristics, such as aTATA-box and other known transcription factor binding site motifs.Identification of such known promoter elements may be used by one ofskill in the art to design variants of the promoter having a similarexpression pattern to the original promoter.

As used herein, the term “enhancer” or “enhancer element” refers to acis-acting transcriptional regulatory element, a.k.a. cis-element, whichconfers an aspect of the overall expression pattern, but is usuallyinsufficient alone to drive transcription, of an operably linkedpolynucleotide sequence. Unlike promoters, enhancer elements do notusually include a transcription start site (TSS) or TATA box. A promotermay naturally comprise one or more enhancer elements that affect thetranscription of an operably linked polynucleotide sequence. An isolatedenhancer element may also be fused to a promoter to produce a chimericpromoter cis-element, which confers an aspect of the overall modulationof gene expression. A promoter or promoter fragment may comprise one ormore enhancer elements that effect the transcription of operably linkedgenes. Many promoter enhancer elements are believed to bind DNA-bindingproteins and/or affect DNA topology, producing local conformations thatselectively allow or restrict access of RNA polymerase to the DNAtemplate or that facilitate selective opening of the double helix at thesite of transcriptional initiation. An enhancer element may function tobind transcription factors that regulate transcription. Some enhancerelements bind more than one transcription factor, and transcriptionfactors may interact with different affinities with more than oneenhancer domain. Enhancer elements may be identified by a number oftechniques, including deletion analysis, i.e., deleting one or morenucleotides from the 5′ end or internal to a promoter; DNA bindingprotein analysis using DNase I footprinting, methylation interference,electrophoresis mobility-shift assays, in vivo genomic footprinting byligation-mediated PCR, and other conventional assays; or by DNA sequencesimilarity analysis using known cis-element motifs or enhancer elementsas a target sequence or target motif with conventional DNA sequencecomparison methods, such as BLAST. The fine structure of an enhancerdomain may be further studied by mutagenesis (or substitution) of one ormore nucleotides or by other conventional methods. Enhancer elements maybe obtained by chemical synthesis or by isolation from regulatoryelements that include such elements, and they may be synthesized withadditional flanking nucleotides that contain useful restriction enzymesites to facilitate subsequence manipulation. Thus, the design,construction, and use of enhancer elements according to the methodsdisclosed herein for modulating the expression of operably linkedtranscribable polynucleotide molecules are encompassed by the presentdisclosure.

As used herein, the term “5′ untranslated flanking region” refers to aDNA molecule isolated from the untranslated 5′ region (5′ UTR) of agenomic copy of a gene and defined generally as a nucleotide segmentbetween the transcription start site (TSS) and the protein codingsequence start site. These sequences, or leaders, may be syntheticallyproduced or manipulated DNA elements. A leader may be used as a 5′regulatory element for modulating expression of an operably linkedtranscribable polynucleotide molecule. Leader molecules may be used witha heterologous promoter or with their native promoter. Promotermolecules of the present disclosure may thus be operably linked to theirnative leader or may be operably linked to a heterologous leader.

As used herein, the term “chimeric” refers to a single DNA moleculeproduced by fusing a first DNA molecule to a second DNA molecule, whereneither first nor second DNA molecule would normally be found in thatconfiguration, i.e. fused to the other. The chimeric DNA molecule isthus a new DNA molecule not otherwise normally found in nature. As usedherein, the term “chimeric promoter” refers to a promoter producedthrough such manipulation of DNA molecules. A chimeric promoter maycombine two or more DNA fragments; an example would be the fusion of apromoter to an enhancer element. Thus, the design, construction, and useof chimeric promoters according to the methods disclosed herein formodulating the expression of operably linked transcribablepolynucleotide molecules are encompassed by the present disclosure.

It is to be understood that nucleotide sequences, located withinintrons, or 3′ of the coding region sequence may also contribute to theregulation of expression of a coding region of interest. Examples ofsuitable introns include, but are not limited to, the maize IVS6 intron,or the maize actin intron. A regulatory element may also include thoseelements located downstream (3′) to the site of transcriptioninitiation, or within transcribed regions, or both. In the context ofthe present disclosure a post-transcriptional regulatory element mayinclude elements that are active following transcription initiation, forexample translational and transcriptional enhancers, translational andtranscriptional repressors, and mRNA stability determinants.

The regulatory elements, or variants or fragments thereof, of thepresent disclosure may be operatively associated with heterologousregulatory elements or promoters in order to modulate the activity ofthe heterologous regulatory element. Such modulation includes enhancingor repressing transcriptional activity of the heterologous regulatoryelement, modulating post-transcriptional events, or either enhancing orrepressing transcriptional activity of the heterologous regulatoryelement and modulating post-transcriptional events. For example, one ormore regulatory elements, or fragments thereof, of the presentdisclosure may be operatively associated with constitutive, inducible,or tissue specific promoters or fragment thereof, to modulate theactivity of such promoters within desired tissues in plant cells.

The disclosure encompasses isolated or recombinant nucleic acidcompositions. An “isolated” or “recombinant” nucleic acid molecule (orDNA) is used herein to refer to a nucleic acid sequence (or DNA) that isno longer in its natural environment, for example in an in vitro or in aheterologous recombinant bacterial or plant host cell. An isolated orrecombinant nucleic acid molecule, or biologically active portionthereof, is substantially free of other cellular material or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized. Anisolated or recombinant nucleic acid is free of sequences (optimallyprotein encoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule maycontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived. TheEVCV regulatory element sequences of the disclosure may be isolated fromthe 5′ untranslated region flanking their respective transcriptioninitiation sites.

Fragments and variants of the disclosed promoter nucleotide sequencesare also encompassed by the present disclosure. In particular, fragmentsand variants of the EVCV regulatory element sequence of SEQ ID NO: 1,SEQ ID NO: 2 or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,may be used in the DNA constructs of the disclosure. As used herein, theterm “fragment” refers to a portion of the nucleic acid sequence.Fragments of an EVCV regulatory element sequence may retain thebiological activity of initiating transcription, more particularlydriving transcription in a constitutive manner. Alternatively, fragmentsof a nucleotide sequence which are useful as hybridization probes maynot necessarily retain biological activity. Fragments of a nucleotidesequence for the EVCV regulatory region may range from at least about 20nucleotides, about 50 nucleotides, about 100 nucleotides, and up to thefull-length nucleotide sequence of the disclosure for the promoterregion of the gene.

A biologically active portion of an EVCV regulatory element may beprepared by isolating a portion of the EVCV regulatory element sequenceof the disclosure, and assessing the promoter activity of the portion.Nucleic acid molecules that are fragments of a EVCV regulatory elementnucleotide sequence comprise at least about 16, 50, 75, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900 or 1000nucleotides, or up to the number of nucleotides present in a full-lengthEVCV regulatory element sequence disclosed herein (for example, 821nucleotides for SEQ ID NO: 2).

For nucleotide sequences, a variant comprises a deletion and/or additionof one or more nucleotides at one or more internal sites within thenative polynucleotide and/or a substitution of one or more nucleotidesat one or more sites in the native polynucleotide. As used herein, a“native” or “genomic” nucleotide sequence comprises a naturallyoccurring nucleotide sequence. For nucleotide sequences, naturallyoccurring variants may be identified with the use of well-knownmolecular biology techniques, as, for example, with polymerase chainreaction (PCR) and hybridization techniques as outlined below. Variantnucleotide sequences also include synthetically derived nucleotidesequences, such as those generated, for example, by using site-directedmutagenesis. Generally, variants of a particular nucleotide sequence ofthe disclosure will have at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to that particular nucleotide sequence asdetermined by sequence alignment programs and parameters describedelsewhere herein. A biologically active variant of a nucleotide sequenceof the disclosure may differ from that sequence by as few as 1-15nucleic acid residues, as few as 1-10, as few as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 nucleic acid residue.

In some embodiments the nucleic acid molecule encoding the regulatoryregion is a “non-genomic nucleic acid sequence”. As used herein a“non-genomic nucleic acid sequence” refers to a nucleic acid moleculethat has one or more changes in the nucleic acid sequence compared tothe native or genomic nucleic acid sequence.

Variant nucleotide sequences also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. With sucha procedure, EVCV regulatory element nucleotide sequences may bemanipulated to create new EVCV regulatory elements. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and may be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the disclosure may be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization, and the like may be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire EVCV regulatoryelement sequence set forth herein or to fragments thereof areencompassed by the present disclosure.

In a PCR approach, oligonucleotide primers may be designed for use inPCR reactions to amplify corresponding DNA sequences from genomic DNAextracted from any plant of interest. Methods for designing PCR primersand PCR cloning are generally known in the art and are disclosed inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinafterSambrook. See also Innis et al., eds. (1990) PCR Protocols: A Guide toMethods and Applications (Academic Press, New York); Innis and Gelfand,eds. (1995) PCR Strategies (Academic Press, New York); and Innis andGelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).Known methods of PCR include, but are not limited to, methods usingpaired primers, nested primers, single specific primers, degenerateprimers, gene-specific primers, vector-specific primers,partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments from a chosen organism. The hybridization probes may belabeled with a detectable group such as P-32 or any other detectablemarker. Thus, for example, probes for hybridization may be made bylabeling synthetic oligonucleotides based on the EVCV regulatory elementsequence of the disclosure. Methods for preparation of probes forhybridization and for construction of genomic libraries are generallyknown in the art and are disclosed in Sambrook.

For example, the entire EVCV regulatory element sequence disclosedherein, or one or more portions thereof, may be used as a probe capableof specifically hybridizing to corresponding EVCV regulatory elementsequences and messenger RNAs. To achieve specific hybridization under avariety of conditions, such probes include sequences that are uniqueamong EVCV regulatory element sequences and are at least about 10nucleotides in length or at least about 20 nucleotides in length. Suchprobes may be used to amplify corresponding EVCV regulatory elementsequences from a chosen plant by PCR. This technique may be used toisolate additional coding sequences from a desired organism, or as adiagnostic assay to determine the presence of coding sequences in anorganism. Hybridization techniques include hybridization screening ofplated DNA libraries (either plaques or colonies; see, for example,Sambrook.

Hybridization of such sequences may be carried out under stringentconditions. The terms “stringent conditions” or “stringent hybridizationconditions” are intended to mean conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a final wash in 0.1×SSC at 60 to 65° C. for a duration of atleast 30 minutes. Duration of hybridization is generally less than about24 hours, usually about 4 to about 12 hours. The duration of the washtime will be at least a length of time sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm (thermal melting point) canbe approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The Tm is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. Tm is reduced by about1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theTm can be decreased 10° C. Generally, stringent conditions are selectedto be about 5° C. lower than the Tm for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the Tm; moderately stringent conditions can utilizea hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the Tm;low stringency conditions can utilize a hybridization and/or wash at 11,12, 13, 14, 15, or 20° C. lower than the Tm. Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See also Sambrook.

Thus, isolated sequences that have constitutive promoter activity andwhich hybridize under stringent conditions to the EVCV regulatoryelement sequences disclosed herein, or to fragments thereof, areencompassed by the present disclosure.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart.

Thus, the determination of percent sequence identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of thedisclosure. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the disclosure. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See the website for the National Center for BiotechnologyInformation. Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. An“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, at least 80%, at least 90%, and at least 95%,compared to a reference sequence using one of the alignment programsdescribed using standard parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the Tm for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C. lower than the Tm, depending upon thedesired degree of stringency as otherwise qualified herein.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the disclosure,provided that these parts comprise the introduced polynucleotides.

The present disclosure may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species include corn (Zea mays), Brassica sp. (e.g., B. napus, B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), Eupatoriums(Eupatorium hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present disclosureinclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent disclosure are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal, and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mung bean, lima bean, fava bean, lentils,chickpea, etc.

As used herein, the term “transcribable polynucleotide molecule” refersto any DNA molecule capable of being transcribed into a RNA molecule,including, but not limited to, those having protein coding sequences andthose having sequences useful for gene suppression. A “transgene” refersto a transcribable polynucleotide molecule heterologous to a host celland/or a transcribable polynucleotide molecule artificially incorporatedinto a host cell's genome.

A regulatory element of the present invention may be operably linked toa transcribable polynucleotide molecule that is heterologous withrespect to the regulatory molecule. As used herein, the term“heterologous” refers to the combination of two or more polynucleotidemolecules when such a combination would not normally be found in nature.For example, the two molecules may be derived from different speciesand/or the two molecules may be derived from different genes, e.g.different genes from the same species or the same genes from differentspecies. A regulatory element is thus heterologous with respect to anoperably linked transcribable polynucleotide molecule if such acombination is not normally found in nature, i.e. that transcribablepolynucleotide molecule is not naturally occurring operably linked incombination with that regulatory element molecule.

The transcribable polynucleotide molecule may generally be any DNAmolecule for which expression of an RNA transcript is desired. Suchexpression of an RNA transcript may result in translation of theresulting mRNA molecule and thus protein expression. Alternatively, atranscribable polynucleotide molecule may be designed to ultimatelycause decreased expression of a specific gene or protein. This may beaccomplished by using a transcribable polynucleotide molecule that isoriented in the antisense direction. One of ordinary skill in the art isfamiliar with using such antisense technology. Briefly, as the antisensetranscribable polynucleotide molecule is transcribed, the RNA producthybridizes to and sequesters a complementary RNA molecule inside thecell. This duplex RNA molecule cannot be translated into a protein bythe cell's translational machinery and is degraded in the cell. Any genemay be negatively regulated in this manner.

Thus, one embodiment of the invention is a regulatory element of thepresent invention, such as those provided as SEQ ID NO: 1-6, operablylinked to a transcribable polynucleotide molecule so as to modulatetranscription of the transcribable polynucleotide molecule at a desiredlevel or in a desired pattern upon introduction of said construct into aplant cell. In one embodiment, the transcribable polynucleotide moleculecomprises a protein-coding region of a gene, and the promoter affectsthe transcription of an RNA molecule that is translated and expressed asa protein product. In another embodiment, the transcribablepolynucleotide molecule comprises an antisense region of a gene, and thepromoter affects the transcription of an antisense RNA molecule or othersimilar inhibitory RNA molecule in order to inhibit expression of aspecific RNA molecule of interest in a target host cell.

Transcribable polynucleotide molecules expressed by the EVCV regulatoryelements of the disclosure may be used for varying the phenotype of aplant. Various changes in phenotype are of interest including modifyingexpression of a gene, altering a plant's pathogen or insect defensemechanism, increasing the plants tolerance to herbicides in a plant,altering root development to respond to environmental stress, modulatingthe plant's response to salt, temperature (hot and cold), drought, andthe like. These results may be achieved by the expression of aheterologous nucleotide sequence of interest comprising an appropriategene product. In specific embodiments, the heterologous nucleotidesequence of interest is an endogenous plant sequence whose expressionlevel is increased in the plant or plant part. Alternatively, theresults may be achieved by providing for a reduction of expression ofone or more endogenous gene products, particularly enzymes,transporters, or cofactors, or by affecting nutrient uptake in theplant. These changes result in a change in phenotype of the transformedplant.

Genes of Agronomic Interest

Transcribable polynucleotide molecules may be genes of agronomicinterest. As used herein, the term “gene of agronomic interest” refersto a transcribable polynucleotide molecule that when expressed in aparticular plant tissue, cell, or cell type provides a desirablecharacteristic associated with plant morphology, physiology, growth,development, yield, product, nutritional profile, disease or pestresistance, and/or environmental or chemical tolerance. Genes ofagronomic interest include, but are not limited to, those encoding ayield protein, a stress resistance protein, a developmental controlprotein, a tissue differentiation protein, a meristem protein, anenvironmentally responsive protein, a senescence protein, a hormoneresponsive protein, an abscission protein, a source protein, a sinkprotein, a flower control protein, a seed protein, an herbicideresistance protein, a disease resistance protein, a fatty acidbiosynthetic enzyme, a tocopherol biosynthetic enzyme, an amino acidbiosynthetic enzyme, a pesticidal protein, or any other agent such as anantisense or RNAi molecule targeting a particular gene for suppression.The product of a gene of agronomic interest may act within the plant inorder to cause an effect upon the plant physiology or metabolism or maybe act as a pesticidal agent in the diet of a pest that feeds on theplant.

In one embodiment, a regulatory element of the present disclosure isincorporated into a construct such that the regulatory is operablylinked to a transcribable polynucleotide molecule that is a gene ofagronomic interest. The expression of the gene of agronomic interest isdesirable in order to confer an agronomically beneficial trait. Abeneficial agronomic trait may be, for example, but not limited to,herbicide tolerance, insect control, modified yield, fungal diseaseresistance, virus resistance, nematode resistance, bacterial diseaseresistance, plant growth and development, starch production, modifiedoils production, high oil production, modified fatty acid content, highprotein production, fruit ripening, enhanced animal and human nutrition,biopolymers, environmental stress resistance, pharmaceutical peptidesand secretable peptides, improved processing traits, improveddigestibility, enzyme production, flavor, nitrogen fixation, hybrid seedproduction, fiber production, and biofuel production.

Insect resistance genes may encode resistance to pests that damage andcause yield drag such as rootworm, cutworm, European corn borer, and thelike. Such genes include, for example, Bacillus thuringiensis toxicprotein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514;5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); and thelike.

Genes encoding disease resistance traits include detoxification genes,such as those which detoxify fumonisin (U.S. Pat. No. 5,792,931);avirulence (avr) and disease resistance (R) genes (Jones et al. (1994)Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos etal. (1994) Cell 78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene),glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example,U.S. Publication No. 20040082770 and WO 03/092360) or other such genesknown in the art. The bar gene encodes resistance to the herbicidebasta, the nptII gene encodes resistance to the antibiotics kanamycinand geneticin, and the ALS-gene mutants encode resistance to theherbicide chlorsulfuron.

Glyphosate resistance is imparted by mutant 5-enolpyruvl-3-phosphikimatesynthase (EPSP) and aroA genes. See, for example, U.S. Pat. No.4,940,835 to Shah et al., which discloses the nucleotide sequence of aform of EPSPS which may confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry et al. also describes genes encoding EPSPS enzymes.See also U.S. Pat. Nos. 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435;5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775;6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; and internationalpublications WO 97/04103; WO 97/04114; WO 00/66746; WO 01/66704; WO00/66747 and WO 00/66748, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene that encodes a glyphosate oxido-reductase enzyme asdescribed more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, whichare incorporated herein by reference for this purpose. In additionglyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.Pat. Nos. 7,714,188 and 7,462,481.

Genes of agronomic interest with regulatory approval are well known toone skilled in the art and can be found at the Center for EnvironmentalRisk Assessment (cera-gmc.org/?action=gm_crop_database, which can beaccessed using the www prefix) and at the International Service for theAcquisition of Agri-Biotech Applicationsisaaa.org/gmapprovaldatabase/default.asp, which can be accessed usingthe www prefix).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like.

Examples of other applicable genes and their associated phenotypeinclude the gene which encodes viral coat protein and/or RNA, or otherviral or plant genes that confer viral resistance; genes that conferfungal resistance; genes that promote yield improvement; and genes thatprovide for resistance to stress, such as cold, dehydration resultingfrom drought, heat and salinity, toxic metal or trace elements, or thelike.

As noted, the heterologous nucleotide sequence operably linked to theEVCV regulatory element disclosed herein may be an antisense sequencefor a targeted gene. Thus the promoter sequences disclosed herein may beoperably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant root.

“RNAi” refers to a series of related techniques to reduce the expressionof genes (See for example U.S. Pat. No. 6,506,559). Older techniquesreferred to by other names are now thought to rely on the samemechanism, but are given different names in the literature. Theseinclude “antisense inhibition,” the production of antisense RNAtranscripts capable of suppressing the expression of the target protein,and “co-suppression” or “sense-suppression,” which refer to theproduction of sense RNA transcripts capable of suppressing theexpression of identical or substantially similar foreign or endogenousgenes (U.S. Pat. No. 5,231,020, incorporated herein by reference). Suchtechniques rely on the use of constructs resulting in the accumulationof double stranded RNA with one strand complementary to the target geneto be silenced. The EVCV regulatory element of the embodiments may beused to drive expression of constructs that will result in RNAinterference including microRNAs and siRNAs.

The regulatory element sequences of the present disclosure, or variantsor fragments thereof, when operably linked to a heterologous nucleotidesequence of interest may drive constitutive expression of theheterologous nucleotide sequence in the plant expressing this construct.A “heterologous nucleotide sequence” is a sequence that is not naturallyoccurring with the regulatory element sequence of the disclosure. Whilethis nucleotide sequence is heterologous to the regulatory elementsequence, it may be homologous, or native, or heterologous, or foreign,to the plant host.

The isolated regulatory element sequences of the present disclosure maybe modified to provide for a range of expression levels of theheterologous nucleotide sequence. Thus, less than the entire regulatoryelement region may be utilized and the ability to drive expression ofthe nucleotide sequence of interest retained. It is recognized thatexpression levels of the mRNA may be altered in different ways withdeletions of portions of the regulatory element sequences. The mRNAexpression levels may be decreased, or alternatively, expression may beincreased as a result of regulatory element deletions if, for example,there is a negative regulatory element (for a repressor) that is removedduring the truncation process. Generally, at least about 20 nucleotidesof an isolated regulatory element sequence will be used to driveexpression of a nucleotide sequence.

It is recognized that to increase transcription levels, enhancers may beutilized in combination with the regulatory element regions of thedisclosure. Enhancers are nucleotide sequences that act to increase theexpression of a regulatory element region. Enhancers are known in theart and include the SV40 enhancer region, the 35S enhancer element, andthe like. Some enhancers are also known to alter normal regulatoryelement expression patterns, for example, by causing a regulatoryelement to drive expression constitutively when without the enhancer,the same regulatory element drives expression only in one specifictissue or a few specific tissues.

Modifications of the isolated regulatory element sequences of thepresent disclosure can provide for a range of expression of theheterologous nucleotide sequence. Thus, they may be modified to be weakpromoters or strong promoters. Generally, a “weak promoter” means apromoter that drives expression of a coding sequence at a low level. A“low level” of expression is intended to mean expression at levels ofabout 1/10,000 transcripts to about 1/100,000 transcripts to about1/500,000 transcripts. Conversely, a strong promoter drives expressionof a coding sequence at a high level, or at about 1/10 transcripts toabout 1/100 transcripts to about 1/1,000 transcripts.

It is recognized that the EVCV regulatory elements of the disclosure maybe used to increase or decrease expression, thereby resulting in achange in phenotype of the transformed plant. This phenotypic changecould further affect an increase or decrease in levels of metal ions intissues of the transformed plant.

The nucleotide sequences disclosed in the present disclosure, as well asvariants and fragments thereof, are useful in the genetic manipulationof a plant. The EVCV regulatory element sequence is useful in thisaspect when operably linked with a heterologous nucleotide sequencewhose expression is to be controlled to achieve a desired phenotypicresponse. The term “operably linked” means that the transcription ortranslation of the heterologous nucleotide sequence is under theinfluence of the regulatory element sequence. In this manner, thenucleotide sequences for the regulatory elements of the disclosure maybe provided in expression cassettes along with heterologous nucleotidesequences of interest for expression in the plant of interest, moreparticularly for expression in the root of the plant.

The regulatory sequences of the embodiments are provided in DNAconstructs for expression in the organism of interest. An “expressioncassette” as used herein means a DNA construct comprising a regulatorysequence of the embodiments operably linked to a heterologouspolynucleotide encoding a polypeptide of interest. Such expressioncassettes will comprise a transcriptional initiation region comprisingone of the regulatory element nucleotide sequences of the presentdisclosure, or variants or fragments thereof, operably linked to theheterologous nucleotide sequence. Such an expression cassette may beprovided with a plurality of restriction sites for insertion of thenucleotide sequence to be under the transcriptional regulation of theregulatory regions. The expression cassette may additionally containselectable marker genes as well as 3′ termination regions.

The expression cassette may include, in the 5′-3′ direction oftranscription, a transcriptional initiation region (i.e., a promoter, orvariant or fragment thereof, of the disclosure), a translationalinitiation region, a heterologous nucleotide sequence of interest, atranslational termination region and, optionally, a transcriptionaltermination region functional in the host organism. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide of theembodiments may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide of theembodiments may be heterologous to the host cell or to each other. Asused herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence beingexpressed, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

The expression cassette comprising the sequences of the presentdisclosure may also contain at least one additional nucleotide sequencefor a gene to be cotransformed into the organism. Alternatively, theadditional sequence(s) can be provided on another expression cassette.

Where appropriate, the nucleotide sequences whose expression is to beunder the control of the constitutive promoter sequence of the presentdisclosure and any additional nucleotide sequence(s) may be optimizedfor increased expression in the transformed plant. That is, thesenucleotide sequences can be synthesized using plant preferred codons forimproved expression. See, for example, Campbell and Gowri (1990) PlantPhysiol. 92:1-11 for a discussion of host-preferred codon usage. Methodsare available in the art for synthesizing plant-preferred genes. See,for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of theheterologous nucleotide sequence may be adjusted to levels average for agiven cellular host, as calculated by reference to known genes expressedin the host cell. When possible, the sequence is modified to avoidpredicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences may act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region) (ElroyStein et al. (1989) Proc. Nat. Acad. Sci. USA 86:6126 6130); potyvirusleaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al.(1986) Virology 154:9 20); MDMV leader (Maize Dwarf Mosaic Virus); humanimmunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90 94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) MolecularBiology of RNA, pages 237 256); and maize chlorotic mottle virus leader(MCMV) (Lommel et al. (1991) Virology 81:382 385). See also Della Cioppaet al. (1987) Plant Physiology 84:965 968. Methods known to enhance mRNAstability may also be utilized, for example, introns, such as the maizeUbiquitin intron (Christensen and Quail (1996) Transgenic Res.5:213-218; Christensen et al. (1992) Plant Molecular Biology 18:675-689)or the maize Adhl intron (Kyozuka et al. (1991) Mol. Gen. Genet.228:40-48; Kyozuka et al. (1990) Maydica 35:353-357), and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, for example,transitions and transversions, may be involved.

Reporter genes or selectable marker genes may be included in theexpression cassettes. Examples of suitable reporter genes known in theart can be found in, for example, Jefferson et al. (1991) in PlantMolecular Biology Manual, ed. Gelvin et al. (Kluwer AcademicPublishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737;Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al. (1995) BioTechniques19:650-655; and Chiu et al. (1996) Current Biology 6:325-330.

Selectable marker genes for selection of transformed cells or tissuesmay include genes that confer antibiotic resistance or resistance toherbicides. Examples of suitable selectable marker genes include, butare not limited to, genes encoding resistance to chloramphenicol(Herrera Estrella et al. (1983) EMBO J. 2:987-992); methotrexate(Herrera Estrella et al. (1983) Nature 303:209-213; Meijer et al. (1991)Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al. (1985) PlantMol. Biol. 5:103-108; and Zhijian et al. (1995) Plant Science108:219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet.210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) TransgenicRes. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker et al. (1988) Science 242:419-423);glyphosate (Shaw et al. (1986) Science 233:478-481; and U.S. applicationSer. Nos. 10/004,357; and 10/427,692); phosphinothricin (DeBlock et al.(1987) EMBO J. 6:2513-2518).

Other genes that could serve utility in the recovery of transgenicevents but might not be required in the final product would include, butare not limited to, examples such as GUS (beta-glucuronidase; Jefferson(1987) Plant Mol. Biol. Rep. 5:387), GFP (green fluorescence protein;Chalfie et al. (1994) Science 263:802), luciferase (Riggs et al. (1987)Nucleic Acids Res. 15(19):8115 and Luehrsen et al. (1992) MethodsEnzymol. 216:397-414) and the maize genes encoding for anthocyaninproduction (Ludwig et al. (1990) Science 247:449).

The expression cassette comprising the EVCV regulatory elements of thepresent disclosure operably linked to a nucleotide sequence of interestmay be used to transform any plant. In this manner, genetically modifiedplants, plant cells, plant tissue, seed, root, and the like may beobtained.

The methods of the disclosure involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the disclosure do not depend on a particularmethod for introducing a sequence into a plant, only that thepolynucleotide or polypeptides gains access to the interior of at leastone cell of the plant. Methods for introducing polynucleotide orpolypeptides into plants are known in the art including, but not limitedto, stable transformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320 334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602 5606),Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055 and Zhao et al., U.S. Pat. No. 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717 2722), and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; 5,932,782; Tomes et al. (1995) in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923926); and Lec1 transformation (WO 00/28058). Also see Weissinger et al.(1988) Ann. Rev. Genet. 22:421 477; Sanford et al. (1987) ParticulateScience and Technology 5:27 37 (onion); Christou et al. (1988) PlantPhysiol. 87:671 674 (soybean); McCabe et al. (1988) Bio/Technology 6:923926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736 740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305 4309(maize); Klein et al. (1988) Biotechnology 6:559 563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440 444 (maize); Fromm et al. (1990) Biotechnology 8:833 839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the DNA constructs comprising the regulatoryelement sequences of the disclosure can be provided to a plant using avariety of transient transformation methods. Such transienttransformation methods include, but are not limited to, viral vectorsystems and the precipitation of the polynucleotide in a manner thatprecludes subsequent release of the DNA. Thus, the transcription fromthe particle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use particles coated with polyethylimine (PEI;Sigma-Aldrich™ #P3143).

In other embodiments, the polynucleotide of the disclosure may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the disclosure within a viral DNA or RNAmolecule. Methods for introducing polynucleotides into plants andexpressing a protein encoded therein, involving viral DNA or RNAmolecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5:209-221; herein incorporated byreference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the disclosure can be contained in transfercassette flanked by two non-identical recombination sites. The transfercassette is introduced into a plant have stably incorporated into itsgenome a target site which is flanked by two non-identical recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present disclosure provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of thedisclosure, for example, an expression cassette of the disclosure,stably incorporated into its genome.

In one embodiment, the EVCV regulatory element sequences may be editedor inserted by genome editing using a CRISPR/Cas9 system.

CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats)(also known as SPIDRs—SPacer Interspersed Direct Repeats) constitute afamily of recently described DNA loci. CRISPR loci consist of short andhighly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to140 times—also referred to as CRISPR-repeats) which are partiallypalindromic. The repeated sequences (usually specific to a species) areinterspaced by variable sequences of constant length (typically 20 to 58by depending on the CRISPR locus (WO2007/025097 published Mar. 1, 2007).

CRISPR loci were first recognized in E. coli (Ishino et al. (1987) J.Bacterial. 169:5429-5433; Nakata et al. (1989) J. Bacterial.171:3553-3556). Similar interspersed short sequence repeats have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (Groenen et al. (1993) Mol. Microbiol.10:1057-1065; Hoe et al. (1999) Emerg. Infect. Dis. 5:254-263; Masepohlet al. (1996) Biochim. Biophys. Acta 1307:26-30; Mojica et al. (1995)Mol. Microbiol. 17:85-93). The CRISPR loci differ from other SSRs by thestructure of the repeats, which have been termed short regularly spacedrepeats (SRSRs) (Janssen et al. (2002) OMICS J. Integ. Biol. 6:23-33;Mojica et al. (2000) Mol. Microbiol. 36:244-246). The repeats are shortelements that occur in clusters, that are always regularly spaced byvariable sequences of constant length (Mojica et al. (2000) Mol.Microbiol. 36:244-246).

Cas gene relates to a gene that is generally coupled, associated orclose to or in the vicinity of flanking CRISPR loci. The terms “Casgene”, “CRISPR-associated (Cas) gene” are used interchangeably herein. Acomprehensive review of the Cas protein family is presented in Haft etal. (2005) Computational Biology, PLoS Comput Biol 1(6): e60.doi:10.1371/journal.pcbi.0010060. As described therein, 41CRISPR-associated (Cas) gene families are described, in addition to thefour previously known gene families. It shows that CRISPR systems belongto different classes, with different repeat patterns, sets of genes, andspecies ranges. The number of Cas genes at a given CRISPR locus can varybetween species.

Cas endonuclease relates to a Cas protein encoded by a Cas gene, whereinsaid Cas protein is capable of introducing a double strand break into aDNA target sequence. The Cas endonuclease is guided by a guidepolynucleotide to recognize and optionally introduce a double strandbreak at a specific target site into the genome of a cell (U.S.Provisional Application No. 62/023,239, filed Jul. 11, 2014). The guidepolynucleotide/Cas endonuclease system includes a complex of a Casendonuclease and a guide polynucleotide that is capable of introducing adouble strand break into a DNA target sequence. The Cas endonucleaseunwinds the DNA duplex in close proximity of the genomic target site andcleaves both DNA strands upon recognition of a target sequence by aguide RNA if a correct protospacer-adjacent motif (PAM) is approximatelyoriented at the 3′ end of the target sequence.

The Cas endonuclease gene can be Cas9 endonuclease, or a functionalfragment thereof, such as but not limited to, Cas9 genes listed in SEQID NOs: 462, 474, 489, 494, 499, 505, and 518 of WO2007/025097 publishedMar. 1, 2007. The Cas endonuclease gene can be a plant, maize or soybeanoptimized Cas9 endonuclease, such as but not limited to a plant codonoptimized streptococcus pyogenes Cas9 gene that can recognize anygenomic sequence of the form N(12-30)NGG. The Cas endonuclease can beintroduced directly into a cell by any method known in the art, forexample, but not limited to transient introduction methods, transfectionand/or topical application.

As used herein, the term “guide RNA” relates to a synthetic fusion oftwo RNA molecules, a crRNA (CRISPR RNA) comprising a variable targetingdomain, and a tracrRNA. In one embodiment, the guide RNA comprises avariable targeting domain of 12 to 30 nucleotide sequences and a RNAfragment that can interact with a Cas endonuclease.

As used herein, the term “guide polynucleotide”, relates to apolynucleotide sequence that can form a complex with a Cas endonucleaseand enables the Cas endonuclease to recognize and optionally cleave aDNA target site (U.S. Provisional Application No. 62/023,239, filed Jul.11, 2014). The guide polynucleotide can be a single molecule or a doublemolecule. The guide polynucleotide sequence can be a RNA sequence, a DNAsequence, or a combination thereof (a RNA-DNA combination sequence).Optionally, the guide polynucleotide can comprise at least onenucleotide, phosphodiester bond or linkage modification such as, but notlimited, to Locked Nucleic Acid (LNA), 5-methyl dC, 2,6-Diaminopurine,2′-Fluoro A, 2′-Fluoro U, 2′-O-Methyl RNA, phosphorothioate bond,linkage to a cholesterol molecule, linkage to a polyethylene glycolmolecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule,or 5′ to 3′ covalent linkage resulting in circularization. A guidepolynucleotide that solely comprises ribonucleic acids is also referredto as a “guide RNA”.

The guide polynucleotide can be a double molecule (also referred to asduplex guide polynucleotide) comprising a first nucleotide sequencedomain (referred to as Variable Targeting domain or VT domain) that iscomplementary to a nucleotide sequence in a target DNA and a secondnucleotide sequence domain (referred to as Cas endonuclease recognitiondomain or CER domain) that interacts with a Cas endonucleasepolypeptide. The CER domain of the double molecule guide polynucleotidecomprises two separate molecules that are hybridized along a region ofcomplementarity. The two separate molecules can be RNA, DNA, and/orRNA-DNA-combination sequences. In some embodiments, the first moleculeof the duplex guide polynucleotide comprising a VT domain linked to aCER domain is referred to as “crDNA” (when composed of a contiguousstretch of DNA nucleotides) or “crRNA” (when composed of a contiguousstretch of RNA nucleotides), or “crDNA-RNA” (when composed of acombination of DNA and RNA nucleotides). The crNucleotide can comprise afragment of the cRNA naturally occurring in Bacteria and Archaea. In oneembodiment, the size of the fragment of the cRNA naturally occurring inBacteria and Archaea that is present in a crNucleotide disclosed hereincan range from, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In someembodiments the second molecule of the duplex guide polynucleotidecomprising a CER domain is referred to as “tracrRNA” (when composed of acontiguous stretch of RNA nucleotides) or “tracrDNA” (when composed of acontiguous stretch of DNA nucleotides) or “tracrDNA-RNA” (when composedof a combination of DNA and RNA nucleotides In one embodiment, the RNAthat guides the RNA/Cas9 endonuclease complex, is a duplexed RNAcomprising a duplex crRNA-tracrRNA.

The guide polynucleotide can also be a single molecule comprising afirst nucleotide sequence domain (referred to as Variable Targetingdomain or VT domain) that is complementary to a nucleotide sequence in atarget DNA and a second nucleotide domain (referred to as Casendonuclease recognition domain or CER domain) that interacts with a Casendonuclease polypeptide. By “domain” it is meant a contiguous stretchof nucleotides that can be RNA, DNA, and/or RNA-DNA-combinationsequence. The VT domain and/or the CER domain of a single guidepolynucleotide can comprise a RNA sequence, a DNA sequence, or aRNA-DNA-combination sequence. In some embodiments the single guidepolynucleotide comprises a crNucleotide (comprising a VT domain linkedto a CER domain) linked to a tracrNucleotide (comprising a CER domain),wherein the linkage is a nucleotide sequence comprising a RNA sequence,a DNA sequence, or a RNA-DNA combination sequence. The single guidepolynucleotide being comprised of sequences from the crNucleotide andtracrNucleotide may be referred to as “single guide RNA” (when composedof a contiguous stretch of RNA nucleotides) or “single guide DNA” (whencomposed of a contiguous stretch of DNA nucleotides) or “single guideRNA-DNA” (when composed of a combination of RNA and DNA nucleotides). Inone embodiment of the disclosure, the single guide RNA comprises a cRNAor cRNA fragment and a tracrRNA or tracrRNA fragment of the type IICRISPR/Cas system that can form a complex with a type II Casendonuclease, wherein said guide RNA/Cas endonuclease complex can directthe Cas endonuclease to a plant genomic target site, enabling the Casendonuclease to introduce a double strand break into the genomic targetsite. One aspect of using a single guide polynucleotide versus a duplexguide polynucleotide is that only one expression cassette needs to bemade to express the single guide polynucleotide.

The term “variable targeting domain” or “VT domain” is usedinterchangeably herein and includes a nucleotide sequence that iscomplementary to one strand (nucleotide sequence) of a double strand DNAtarget site. The % complementation between the first nucleotide sequencedomain (VT domain) and the target sequence can be at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable target domain can beat least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 nucleotides in length. In some embodiments, the variabletargeting domain comprises a contiguous stretch of 12 to 30 nucleotides.The variable targeting domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence, or anycombination thereof.

The term “Cas endonuclease recognition domain” or “CER domain” of aguide polynucleotide is used interchangeably herein and includes anucleotide sequence (such as a second nucleotide sequence domain of aguide polynucleotide), that interacts with a Cas endonucleasepolypeptide. The CER domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence (see forexample modifications described herein), or any combination thereof.

The nucleotide sequence linking the crNucleotide and the tracrNucleotideof a single guide polynucleotide can comprise a RNA sequence, a DNAsequence, or a RNA-DNA combination sequence. In one embodiment, thenucleotide sequence linking the crNucleotide and the tracrNucleotide ofa single guide polynucleotide can be at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100 nucleotides in length. In another embodiment, the nucleotidesequence linking the crNucleotide and the tracrNucleotide of a singleguide polynucleotide can comprise a tetraloop sequence, such as, but notlimiting to a GAAA tetraloop sequence.

Nucleotide sequence modification of the guide polynucleotide, VT domainand/or CER domain can be selected from, but not limited to, the groupconsisting of a 5′ cap, a 3′ polyadenylated tail, a riboswitch sequence,a stability control sequence, a sequence that forms a dsRNA duplex, amodification or sequence that targets the guide polynucleotide to asubcellular location, a modification or sequence that provides fortracking, a modification or sequence that provides a binding site forproteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a2,6-Diaminopurine nucleotide, a 2′-Fluoro A nucleotide, a 2′-Fluoro Unucleotide; a 2′-O-Methyl RNA nucleotide, a phosphorothioate bond,linkage to a cholesterol molecule, linkage to a polyethylene glycolmolecule, linkage to a spacer 18 molecule, a 5′ to 3′ covalent linkage,or any combination thereof. These modifications can result in at leastone additional beneficial feature, wherein the additional beneficialfeature is selected from the group of a modified or regulated stability,a subcellular targeting, tracking, a fluorescent label, a binding sitefor a protein or protein complex, modified binding affinity tocomplementary target sequence, modified resistance to cellulardegradation, and increased cellular permeability.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this disclosure pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

The above description of various illustrated embodiments of thedisclosure is not intended to be exhaustive or to limit the scope to theprecise form disclosed. While specific embodiments of an examples aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. The teachings providedherein can be applied to other purposed, other than the examplesdescribed above. Numerous modifications and variations are possible inlight of the above teachings and, therefore, are within the scope of theappended claims.

These and other changes may be made in light of the above detaileddescription. In general, in the following claims, the terms used shouldnot be construed to limit the scope to the specific embodimentsdisclosed in the specification and the claims.

Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.), but someexperimental errors and deviations should be allowed for. Unlessotherwise indicated, parts are parts by weight; molecular weight isaverage molecular weight; temperature is in degrees centigrade; andpressure is at or near atmospheric.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1: Eupatorium Vein Clearing Virus RegulatoryElement Sequences

The regulatory element of SEQ ID NO: 1 was obtained through a search ofGenBank Genomes for viral genomes that had been sequenced and belongedto the Caulimoviridae virus family. The search was initiated based onthe well-known Cauliflower Mosaic Virus 35S (CaMV35S) promoter. Itdrives constitutive expression of heterologous genes in most tissues ofmost plants. Other regulatory elements from this virus family, such asthe Figwort Mosaic Virus 34S promoter also direct constitutive-likeexpression in plants. Therefore, additional regulatory elements derivedfrom the Caulimoviridae virus family also may drive constitutiveexpression in plants. The region of the genome found in what is calledthe Large Intergenic Region (LIR) generally contains the regulatorysequences necessary for promoter function in plants.

The Eupatorium Vein Clearing Virus (EVCV) genome has an LIR, so thisregion was targeted for functional analysis of regulatory elements. Onefull-length sequence was selected to be tested in plants. The sequenceconsists of 1104 bp (set forth in SEQ ID NO: 1) and has a putative TATAstarting approximately box 88 bp upstream of the 3′ end of the sequence.The entire 1104 bp sequence is referred to as the EVCV FL (full-length)regulatory element or EVCV FL. Deleting segments of the 5′ end of thefull-length regulatory element may alter the expression pattern andprovide insight into important sequence markers in the regulatoryregion. Second, third, fourth, fifth and sixth sequences are a truncatedversion of the full-length regulatory element (See FIG. 2; SEQ ID NO:1). EVCV TR1, EVCV TR2, EVCV TR3, EVCV TR4, and EVCV TR5 regulatoryelements respectively consist of 821 bp, 514 bp, 431 bp, 283 bp, and 116bp and contain the 3′ end of the full-length promoter (SEQ ID NO: 2-6).

Example 2: Expression and Deletion Analysis of the EVCV RegulatoryElement

EVCV FL was operably linked to the first intron of the maize alcoholdehydrogenase gene 1 (ADH1 intron1) and the β-glucuronidase (GUS) gene,in an expression vector, to test whether the synthesized DNA fragmentwould direct expression (SEQ ID NO: 1). ADH1 intron1 was included forthe purpose of increased expression as it has been shown that in cerealplant cells the expression of transgenes is enhanced by the presence ofsome 5′ proximal introns (See Callis et al. (1987) Genes and Development1: 1183-1200; Kyozuka et al. (1990) Maydica 35:353-357).

The Ubi-1 promoter and intron from Zea mays were operably linked to theGUS gene so that it could be used to compare the expression pattern andexpression levels of the EVCV regulatory elements. The Ubi-1 promoter isa strong constitutive promoter in most tissues of Zea mays.

Regulatory elements are a collection of sequence motifs that worktogether to bind transcription factors that result in the spatial,temporal, and quantitative expression characteristics of a promoter.Understanding the architecture and the positioning of these motifsenhances knowledge pertaining to the regulatory element. Segmentaldeletion analysis is an important tool that may be used to begin toidentify regions of a regulatory element that contain functionallyimportant motifs. The removal of segments from the 5′ end may change thespatial, temporal, and/or quantitative expression patterns directed bythe regulatory element. The regions that result in a change may then bestudied more closely to evaluate the sequences and their interactionwith cis and trans factors. The truncations may also identify a minimalfunctional sequence.

The restriction endonuclease recognition sites, SpeI, PvuII, SnaBI,HindIII and DraI were used to remove five sequence regions in EVCV FLranging in size from about 307 to 83 bp. EVCV TR1, EVCV TR2, EVCV TR3,EVCV TR4, EVCV TR5 were operably linked to ADH1 intron1 and the GUS genein an expression vector to test the expression potential of thesynthesized DNA fragments (SEQ ID NO: 2-6).

Stable transformed plants were created using Agrobacterium protocols(detailed in Example 3) to allow for the characterization of promoteractivity, including spatial and quantitative expression directed by thedifferent regulatory elements. Plants grown to V5/6 stage undergreenhouse conditions were sampled for leaf and root material toevaluate expression changes via histochemical GUS staining analysis andquantitative fluorometric assays. Vegetative growth stages aredetermined by the number of collared leaves on the plant. Therefore, aplant at V5 stage has 5 fully collared leaves. The plants were thenallowed to grow to R1-R2 stage, a point when silks emerge outside thehusk (R1 and just start to dry out (R2). Tissues, that includedreproductive tissues, were collected and analyzed for expression (Table1).

TABLE 1 Plant Expression Results for the EVCV Promoter (with ADH1intron1 and GUS) V5-V6 R1-R2 Leaf Root Stalk Tassel Husk Silk PollenEVCV FL 3 3.5 3 3 5 2.5 <0.1 EVCV TR1 3 3.5 3 3 4.75 2.5 <0.1 EVCV TR2 33.5 3 3 4.75 2.5 <0.1 EVCV TR3 3 3.5 3 3 4.50 2.5 <0.2 EVCV TR4 3 3.5 22.5 4.25 2.5 <0.5 EVCV TR5 0 0 0 0 0 0 <1 Ubi-1 3 3 3 3 5 2 3untransformed 0 0 0 0 0 0 0 (negative control) Data expressed on a 0-6scale with the maize Ubi-1 promoter directed expression as a comparator.

The EVCV FL (SEQ ID NO: 1) regulatory element was operably linked toADH1 intron1 and an insecticidal gene (abbreviated IG2) to testexpression. The Ubi-1 promoter and intron driving the expression of theIG2 insecticidal gene was used for comparison in the analysis. Stabletransformed plants were created using Agrobacterium protocols (detailedin Example 3) and allowed to grow to a developmental stage of V5/6 whenleaves were sampled. The plants were then allowed to grow to R1-R2 stagewhen stalk and pollen samples were taken. Kernels were sampled when theyreached maturity.

Results from enzyme-linked immunosorbent assays (ELISA) against IG2showed the EVCV FL regulatory element (SEQ ID NO: 1) directed expressionin leaf, stalk, and kernel tissues (Table 2). Expression levels werecomparable to the Ubi-1 promoter and its intron in leaf and stalktissues. In kernels, EVCV FL (SEQ ID NO: 1) directed expression waslower than Ubi-1 and in pollen expression was essentially very low.

The expression data were supported by results from insect consumptionassays. Feeding insects dissected plant tissues provides a rapidassessment of protein expression, as sufficient levels are needed toprotect the tissue from the insects. Insufficient expression will resultin feeding damage. Both EVCV FL (SEQ ID NO: 1) and Ubi-1 plantsdemonstrated levels of expression that protected leaf and silk tissueagainst insect damage. Tissues from negative control plants that did nothave the IG2 gene were consumed.

TABLE 2 Plant Expression Results for the EVCV Regulatory element (withADH1 intron1 and IG2) V5-V6 R1-R2 Maturity Leaf Stalk Pollen KernelsEVCV FL 2 3 <0.1 <0.75 Ubi-1 2 3 3 2 untransformed 0 0 0 0 (negativecontrol) Data expressed on a 0-6 scale with the maize Ubi-1 promoterrepresenting a median value.

Example 3: Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with a regulatoryelement sequence of the disclosure, the method of Zhao was employed(U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; thecontents of which are hereby incorporated by reference). Briefly,immature embryos were isolated from maize and the embryos contacted witha suspension of Agrobacterium under conditions whereby the bacteria werecapable of transferring the regulatory element sequence of thedisclosure to at least one cell of at least one of the immature embryos(step 1: the infection step). In this step the immature embryos wereimmersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos were co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). The immature embryoswere cultured on solid medium following the infection step. Followingthe co-cultivation period an optional “resting” step was performed. Inthis resting step, the embryos were incubated in the presence of atleast one antibiotic known to inhibit the growth of Agrobacteriumwithout the addition of a selective agent for plant transformants (step3: resting step). Next, inoculated embryos were cultured on mediumcontaining a selective agent and growing transformed calli wererecovered (step 4: the selection step). Plantlets were regenerated fromthe calli (step 5: the regeneration step) prior to transfer to thegreenhouse.

Example 4: Expression Analysis of the EVCV Regulatory Element in Canola

The EVCV FL promoter (SEQ ID NO: 1) was tested in canola using the GUSgene as a reporter. Biolistic bombardment transient assays were used toprovide an initial assessment of performance. The number of GUS stainingfoci and the intensity of staining was compared to the Arabidopsisubiqutin-10 promoter (AtUBQ10), a strong promoter in canola tissues.Performance of the EVCV FL (SEQ ID NO: 1) promoter was comparable to theAtUBQ10 in the number of foci and the intensity of GUS staining.

Transgenic canola plants were generated with both the EVCV FL:GUS andAtUBQ10:GUS vectors. Histochemical staining of EVCV FL:GUS events showedhigh expression in leaves and floral organs. EVCV FL directed expressionwas also observed in pollen and siliques. When compared againsthistochemically stained tissue from AtUBQ10:GUS plants, EVCV FL wascomparable in leaves and floral organs. In pollen, EVCV FL directedexpression was weaker than AtUBQ10, with about 10-15% of the pollengrains staining. Almost all of the AtUBQ10 grains stained darkly.

TABLE 3 Expression Results for the EVCV FL Promoter in Canola TransientFloral assay Leaf organs Pollen EVCV FL 3 3 3 1 AtUBQ10 3 3 3 3Untransformed 0 0 0 0 (negative control) Data expressed on a 0-3 scalethree indicating strong expression.

What is claimed is:
 1. A recombinant nucleic acid molecule; comprising aregulatory element selected from the group consisting of: (a) apolynucleotide having at least 85 percent sequence identity to thenucleic acid sequence of SEQ ID NO: 1, (b) the polynucleotide of SEQ IDNO: 1, and (c) a fragment of SEQ ID NO:
 1. 2. The recombinant nucleicacid molecule of claim 1, wherein the polynucleotide has at least 90percent sequence identity to the nucleic acid sequence of SEQ ID NO: 1.3. The recombinant nucleic acid molecule of claim 1, wherein thepolynucleotide has at least 95 percent sequence identity to a nucleicacid sequence of SEQ ID NO:
 1. 4. The recombinant nucleic acid moleculeof claim 1, wherein the polynucleotide comprises a regulatory elementselected from the group consisting of; (a) a polynucleotide having atleast 85 percent sequence identity to the nucleic acid sequence of SEQID NO: 2, (b) the polynucleotide of SEQ ID NO: 2, and (c) a fragment ofSEQ ID NO:
 2. 5. The recombinant nucleic acid molecule of claim 1,wherein the polynucleotide comprises a regulatory element selected fromthe group consisting of; (a) a polynucleotide having at least 85 percentsequence identity to the nucleic acid sequence of SEQ ID NO: 3, (b) thepolynucleotide of SEQ ID NO: 3, and (c) a fragment of SEQ ID NO:
 3. 6.The recombinant nucleic acid molecule of claim 1, wherein thepolynucleotide comprises a regulatory element selected from the groupconsisting of; (a) a polynucleotide having at least 85 percent sequenceidentity to the nucleic acid sequence of SEQ ID NO: 4, (b) thepolynucleotide of SEQ ID NO: 4, and (c) a fragment of SEQ ID NO:
 4. 7.The recombinant nucleic acid molecule of claim 1, wherein thepolynucleotide comprises a regulatory element selected from the groupconsisting of; (a) a polynucleotide having at least 85 percent sequenceidentity to the nucleic acid sequence of SEQ ID NO: 5, (b) thepolynucleotide of SEQ ID NO: 5, and (c) a fragment of SEQ ID NO:
 5. 8.The recombinant nucleic acid molecule of claim 1, wherein thepolynucleotide comprises a regulatory element selected from the groupconsisting of; (a) a polynucleotide having at least 85 percent sequenceidentity to the nucleic acid sequence of SEQ ID NO: 6, (b) thepolynucleotide of SEQ ID NO, and (c) a fragment of SEQ ID NO:
 6. 9. ADNA construct; comprising (a) a regulatory element selected from thegroup consisting of: (i) a polynucleotide with at least 85 percentsequence identity to the nucleic acid sequence of SEQ ID NO: 1, (ii) thepolynucleotide of SEQ ID NO: 1, and (ii) a fragment of SEQ ID NO: 1; and(b) a heterologous transcribable polynucleotide molecule operably linkedto the regulatory element.
 10. The DNA construct of claim 9, wherein thepolynucleotide has at least 90 percent sequence identity to the nucleicacid sequence of SEQ ID NO:
 1. 11. The DNA construct of claim 9, whereinthe polynucleotide has at least 95 percent sequence identity to thenucleic acid sequence of SEQ ID NO:
 1. 12. The DNA construct of claim 9;comprising (a) regulatory element selected from the group consisting of:(i) a polynucleotide with at least 85 percent sequence identity to thenucleic acid sequence of SEQ ID NO: 2, (ii) the polynucleotide of SEQ IDNO: 2, and (ii) a fragment of SEQ ID NO: 2; and (b) a heterologoustranscribable polynucleotide molecule, operably linked to the regulatoryelement.
 13. The DNA construct of claim 9; comprising (a) regulatoryelement selected from the group consisting of: (i) a polynucleotide withat least 85 percent sequence identity to the nucleic acid sequence ofSEQ ID NO: 3, (ii) the polynucleotide of SEQ ID NO: 3, and (ii) afragment of SEQ ID NO: 3; and (b) a heterologous transcribablepolynucleotide molecule operably linked to the regulatory element. 14.The DNA construct of claim 9; comprising (a) regulatory element selectedfrom the group consisting of: (i) a polynucleotide with at least 85percent sequence identity to the nucleic acid sequence of SEQ ID NO: 4,(ii) the polynucleotide of SEQ ID NO: 4, and (ii) a fragment of SEQ IDNO: 4; and (b) a heterologous transcribable polynucleotide moleculeoperably linked to the regulatory element.
 15. The DNA construct ofclaim 9; comprising (a) regulatory element selected from the groupconsisting of: (i) a polynucleotide with at least 85 percent sequenceidentity to the nucleic acid sequence of SEQ ID NO: 5, (ii) thepolynucleotide of SEQ ID NO: 5, and (ii) a fragment of SEQ ID NO: 5; and(b) a heterologous transcribable polynucleotide molecule operably linkedto the regulatory element.
 16. The DNA construct of claim 9; comprising(a) regulatory element selected from the group consisting of: (i) apolynucleotide with at least 85 percent sequence identity to the nucleicacid sequence of SEQ ID NO: 6, (ii) the polynucleotide of SEQ ID NO: 6,and (ii) a fragment of SEQ ID NO: 6; and (b) a heterologoustranscribable polynucleotide molecule operably linked to the regulatoryelement.
 17. The DNA construct of claim 9, wherein the heterologoustranscribable polynucleotide molecule is a gene of agronomic interest.18. The DNA construct of claim 17, wherein the heterologoustranscribable polynucleotide molecule is a gene capable of providingherbicide resistance in plants.
 19. The DNA construct of claim 17,wherein the heterologous transcribable polynucleotide molecule is a genecapable of providing plant pest control in plants.
 20. A transgenicplant or plant cell stably transformed with the nucleic acid molecule ofclaim
 1. 21. The transgenic plant or plant cell of claim 20, wherein thetransgenic plant is a monocotyledon plant cell.
 22. The transgenic plantor plant cell of claim 20, wherein the transgenic plant is a dicotyledonplant cell.
 23. A transgenic plant or plant cell stably transformed withthe DNA construct of claim
 9. 24. A plant part of the transgenic plantof claim 22, wherein the plant part comprises the DNA construct.
 25. Aseed of the transgenic plant of claim 21, wherein the seed comprises theDNA construct.